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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
  • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
  • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin

Definitions

• the present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health More particularly, the present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an AveC gene product, which can be used to modulate the ratio of class 2 1 avermectins produced by fermentation of cultures of Streptomyces avermitilis, and to compositions and methods for screening for such polynucleotide molecules The present invention further relates to vectors, transformed host cells, and novel mutant strains of S avermitilis in which the aveC gene has been mutated so as to modulate the ratio of class 2 1 avermectins produced
• Avermectins Streptomyces species produce a wide variety of secondary metabolites, including the avermectins which comprise a series of eight related sixteen-membered macrocyclic lactones having potent anthelmintic and insecticidal activity
• the eight distinct but closely related compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b
• the "a” series of compounds refers to the natural avermectin where the substituent at the C25 position is (S)- sec-butyl, and the "b” series refers to those compounds where the substituent at the C25 position is isopropyl
• the designations "A” and “B” refer to avermectins where the substituent at the C5 position is methoxy and hydroxy, respectively
• the numeral "1" refers to avermectins where a double bond is present at the C22
• ave genes Genes involved in avermectin biosynthesis (ave genes), like the genes required for biosynthesis of other Streptomyces secondary metabolites (e.g., PKS), are found clustered on the chromosome. A number of ave genes have been successfully cloned using vectors to complement S. avermitilis mutants blocked in avermectin biosynthesis. The cloning of such genes is described in U.S. Patent 5,252,474. In addition, Ikeda et al., 1995, J. Antibiot.
• ivermectin a potent anthelmintic compound, can be produced chemically from avermectin
• B2a such a single component producer of avermectin B2a is considered particularly useful for commercial production of ivermectin.
• the present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of S. avermitilis or a substantial portion thereof, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the S. avermitilis chromosome.
• the isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucleotide sequence of the aveC ORF of FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.
• the present invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO:1) or substantial portion thereof.
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the ammo acid sequence of FIGURE 1 (SEQ ID NO 2) or substantial portion thereof
• the present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an AveC homolog gene product
• the isolated polynucleotide molecule comprises a nucleotide sequence encoding the AveC homolog gene product from S hygroscopicus, which homolog gene product comprises the ammo acid sequence of SEQ ID NO 4 or a substantial portion thereof
• the isolated polynucleotide molecule of the present invention that encodes the S hygroscopicus AveC homolog gene product comprises the nucleotide sequence of SEQ ID NO 3 or a substantial portion thereof
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S hygroscopicus nucleotide sequence of SEQ ID NO 3
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the S hygroscopicus AveC homolog gene product having the ammo acid sequence of SEQ ID NO 4
• the present invention further provides oligonucleotides that hybridize to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO 1) or SEQ ID NO 3, or to a polynucleotide molecule having a nucleotide sequence which is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO 1) or SEQ ID NO 3
• the present invention further provides recombinant cloning vectors and expression vectors, that are useful in cloning or expressing a polynucleotide of the present invention, including polynucleotide molecules comprising the aveC ORF of S avermitilis or an aveC homolog ORF
• the present invention provides plasmid pSE186 (ATCC 209604), which comprises the entire ORF of the aveC gene of S avermitilis
• the present invention further provides transformed host cells comprising a polynucleotide molecule or
• the present invention further provides a recombinantly expressed AveC gene product or AveC homolog gene product, or a substantial portion thereof, that has been substantially purified or isolated, as well as homologs thereof
• the present invention further provides a method for producing a recombinant AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product or AveC homolog gene product, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product or AveC homolog gene product, and recovering the AveC gene product or AveC homolog gene product from the cell culture
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that
• the present invention provides methods for identifying mutations of the aveC ORF of S avermitilis capable of altering the ratio and/or amount of avermectins produced
• a method for identifying mutations of the aveC ORF capable of altering the class 2 1 ratio of avermectins produced comprising (a) determining the class 2 1 ratio of avermectins produced by cells of a strain of S avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product has been introduced and is being expressed, (b) determining the class 2 1 ratio of avermectins produced by cells of the same strain of S avermitilis as in step (a) but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE
• avermitilis cells of step (a) to the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step (b); such that if the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step (a) is different from the class 2:1 ratio of avermectins produced by the S. avermitilis cells of step (b), then a mutation of the aveC ORF capable of altering the class 2:1 ratio of avermectins has been identified. In a preferred embodiment, the class 2:1 ratio of avermectins is reduced by the mutation.
• the present invention provides a method for identifying mutations of the aveC ORF or genetic constructs comprising the aveC ORF capable of altering the amount of avermectins produced, comprising: (a) determining the amount of avermectins produced by cells of a strain of S.
• avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product has been introduced and is being expressed; (b) determining the amount of avermectins produced by cells of the same strain of S.
• avermitilis as in step (a) but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO:1) or a nucleotide sequence that is homologous thereto; and (c) comparing the amount of avermectins produced by the S. avermitilis cells of step (a) to the amount of avermectins produced by the S. avermitilis cells of step (b); such that if the amount of avermectins produced by the S. avermitilis cells of step (a) is different from the amount of avermectins produced by the S.
• avermitilis cells of step (b) then a mutation of the aveC ORF or a genetic construct capable of altering the amount of avermectins has been identified.
• the amount of avermectins produced is increased by the mutation.
• the present invention further provides recombinant vectors that are useful for making novel strains of S. avermitilis having altered avermectin production.
• the present invention provides vectors that can be used to target any of the polynucleotide molecules comprising the mutated nucleotide sequences of the present invention to the site of the aveC gene of the S. avermitilis chromosome to either insert into or replace the aveC ORF or a portion thereof by homologous recombination.
• a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention provided herewith can also function to modulate avermectin biosynthesis when inserted into the S. avermitilis chromosome at a site other than at the aveC gene, or when maintained episomally in S. avermitilis cells.
• the present invention also provides vectors comprising a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention, which vectors can be used to insert the polynucleotide molecule at a site in the S.
• avermitilis chromosome other than at the aveC gene, or to be maintained episomally.
• the present invention provides gene replacement vectors that can be used to insert a mutated aveC allele into the S. avermitilis chromosome to generate novel strains of cells that produce avermectins in a reduced class 2:1 ratio compared to the cells of the same strain which instead express only the wild-type aveC allele.
• the present invention further provides methods for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce altered ratios and/or amounts of avermectins compared to cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele.
• the present invention provides a method for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce an altered class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising transforming cells of a strain of S.
• avermitilis with a vector that carries a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered class 2:1 ratio compared to the class 2:1 ratio produced by cells of the strain that instead express the wild-type aveC allele.
• the class 2:1 ratio of avermectins produced is reduced in cells of the novel strain.
• the present invention provides a method for making novel strains of S. avermitilis comprising cells that produce altered amounts of avermectin, comprising transforming cells of a strain of S. avermitilis with a vector that carries a mutated aveC allele or a genetic construct comprising the aveC allele, the expression of which results in an altered amount of avermectins produced by cells of a strain of S.
• avermitilis expressing the mutated aveC allele or genetic construct as compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only the wild-type aveC allele.
• the amount of avermectins produced is increased in cells of the novel strain.
• the present invention provides a method for making novel strains of S. avermitilis, the cells of which comprise an inactivated aveC allele,
• the present invention further provides novel strains of S. avermitilis comprising ceils that have been transformed with any of the polynucleotide molecules or vectors comprising a mutated nucleotide sequence of the present invention.
• the present invention provides novel strains of S. avermitilis comprising cells which express a mutated aveC allele in place of, or in addition to, the wild-type aveC allele, wherein the cells of the novel strain produce avermectins in an altered class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele.
• the cells of the novel strain produce avermectins in a reduced class 2:1 ratio compared to cells of the same strain that instead express only the wild-type aveC allele.
• Such novel strains are useful in the large-scale production of commercially desirable avermectins such as doramectin.
• the present invention provides novel strains of S.
• avermitilis comprising cells which express a mutated aveC allele, or a genetic construct comprising the aveC allele, in place of, or in addition to, the wild-type aveC allele, which results in the production by the cells of an altered amount of avermectins compared to the amount of avermectins produced by cells of the same strain that instead express only the wild-type aveC allele.
• the novel cells produce an increased amount of avermectins.
• the present invention provides novel strains of S. avermitilis comprising cells in which the aveC gene has been inactivated.
• Such strains are useful both for the different spectrum of avermectins that they produce compared to the wild- type strain, and in complementation screening assays as described herein, to determine whether targeted or random mutagenesis of the aveC gene affects avermectin production.
• the present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S. avermitilis, which cells express a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain which do not express the mutated aveC allele but instead express only the wild- type aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture.
• the class 2:1 ratio of avermectins produced by cells expressing the mutation is
• the present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S avermitilis, which cells express a mutated aveC allele or a genetic construct comprising an aveC allele that results in the production of an altered amount of avermectins produced by cells of a strain of S avermitilis expressing the mutated aveC allele or genetic construct compared to cells of the same strain which do not express the mutated aveC allele or genetic construct but instead express only the wild-type aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture
• the amount of avermectins produced by cells expressing the mutation or genetic construct is increased
• the present invention further provides a novel composition of avermectins produced by a strain of S avermitilis expressing a mutated aveC allele of the present invention, wherein the avermectins are produced in a reduced class 2 1 ratio as compared to the class 2 1 ratio of avermectins produced by cells of the same strain of S avermitilis that do not express the mutated aveC allele but instead express only the wild-type aveC allele
• the novel avermectin composition can be present as produced in fermentation culture fluid, or can be harvested therefrom, and can be partially or substantially purified therefrom 4.
• FIGURE 1 DNA sequence (SEQ ID NO 1) comprising the S avermitilis aveC ORF, and deduced ammo acid sequence (SEQ ID NO 2)
• FIGURE 2 Plasmid vector pSE186 (ATCC 209604) comprising the entire ORF of the aveC gene of S avermitilis
• FIGURE 3 Gene replacement vector pSE180 (ATCC 209605) comprising the ermE gene of Sacc erythraea inserted into the aveC ORF of S avermitilis
• FIGURE 4 BamHI restriction map of the avermectin polyketide synthase gene cluster from S avermitilis with five overlapping cosmid clones identified (; e , pSE65, pSE66, pSE67, pSE68, pSE69) The relationship of pSE118 and pSE119 is also indicated FIGURE 5 HPLC analysis of fermentation products produced by S avermitilis strains Peak quantitation was performed by comparison to standard quantities of cyclohexyl B1 Cyclohexyl B2 retention time was 7 4-7 7 mm, cyclohexyl B1 retention time was 11 9-12 3 mm
• FIG 5A S avermitilis strain SE180-11 with an inactivated aveC ORF
• FIG 5B S avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604)
• FIG 5C S avermitilis strain SE180-11 transformed with pSE187
• FIG 5D S aver
• FIGURE 6 Comparison of deduced ammo acid sequences encoded by the aveC ORF of S avermitilis (SEQ ID NO 2), an aveC homolog partial ORF from S griseochromogenes (SEQ ID NO 5), and the aveC homolog ORF from S hygroscopicus (SEQ ID NO 4)
• the valme residue in bold is the putative start site for the protein conserveed residues are shown in capital letters for homology in all three sequences and in lower case letters for homology in 2 of the 3 sequences
• the ammo acid sequences contain approximately 50% sequence identity
• FIGURE 7 Hybrid plasmid construct containing a 564 bp BsaA ⁇ /Kpn ⁇ fragment from the S hygroscopicus aveC homolog gene inserted into the BsaA ⁇ IKpn ⁇ site in the S avermitilis aveC ORF
• the present invention relates to the identification and characterization of polynucleotide molecules having nucleotide sequences that encode the AveC gene product from Streptomyces avermitilis, the construction of novel strains of S avermitilis that can be used to screen mutated AveC gene products for their effect on avermectin production, and the discovery that certain mutated AveC gene products can reduce the ratio of B2 B1 avermectins produced by S avermitilis
• the invention is described in the sections below for a polynucleotide molecule having either a nucleotide sequence that is the same as the S avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO 1), and for polynucleotides molecules having mutated nucleotide sequence
• the present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of S avermitilis or a substantial portion thereof, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the S avermitilis chromosome
• the isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the S avermitilis AveC gene product-encoding
• a "substantial portion" of an isolated polynucleotide molecule comprising a nucleotide sequence encoding the S. avermitilis AveC gene product means an isolated polynucleotide molecule comprising at least about 70% of the complete aveC ORF sequence shown in FIGURE 1 (SEQ ID NO:1 ), and that encodes a functionally equivalent AveC gene product.
• a "functionally equivalent” AveC gene product is defined as a gene product that, when expressed in S.
• avermitilis strain ATCC 53692 in which the native aveC allele has been inactivated results in the production of substantially the same ratio and amount of avermectins as produced by S. avermitilis strain ATCC 53692 which instead expresses only the wild-type, functional aveC allele native to S. avermitilis strain ATCC 53692.
• the isolated polynucleotide molecule f the present invention can further comprise nucleotide sequences that naturally flank the aveC gene in situ in S. avermitilis, such as those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1).
• polynucleotide molecule As used herein, the terms “polynucleotide molecule,” “polynucleotide sequence,” “coding sequence,” “open-reading frame,” and “ORF” are intended to refer to both DNA and RNA molecules, which can either be single-stranded or double-stranded, and that can be transcribed and translated (DNA), or translated (RNA), into an AveC gene product or, as described below, into an AveC homolog gene product, or into a polypeptide that is homologous to an AveC gene product or AveC homolog gene product in an appropriate host cell expression system when placed under the control of appropriate regulatory elements.
• a coding sequence can include but is not limited to prokaryotic sequences, cDNA sequences, genomic DNA sequences, and chemically synthesized DNA and RNA sequences.
• the nucleotide sequence shown in FIGURE 1 (SEQ ID NO:1) comprises four different
• GTG codons at bp positions 42, 174, 177 and 180 are constructed to help define which of these codons could function in the aveC ORF as start sites for protein expression. Deletion of the first GTG site at bp 42 did not eliminate AveC activity. Additional deletion of all of the GTG codons at bp positions 174, 177 and 180 together eliminated AveC activity, indicating that this region is necessary for protein expression.
• the present invention thus encompasses variable length aveC ORFs that initiate translation at any of the GTG sites located at bp 174, 177 or 180 bp, as presented in FIGURE 1 (SEQ ID NO:1), and corresponding polypeptides for each.
• the present invention further provides a polynucleotide molecule having a nucleotide sequence that is homologous to the S avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO 1) or substantial portion thereof
• the term "homologous" when used to refer to a polynucleotide molecule that is homologous to an S avermitilis AveC gene product-encoding sequence means a polynucleotide molecule having a nucleotide sequence (a) that encodes the same AveC gene product as the S avermitilis AveC gene product- encoding sequence of plasmid pSE186 (ATCC 209604), or that encodes the same AveC gene product as the nucleotide sequence of the aveC ORF presented in FIGURE 1 (SEQ ID NO 1), but that includes one or more
• an AveC gene product and potential functional equivalents thereof can be determined through HPLC analysis of fermentation products, as described in the examples below
• Polynucleotide molecules having nucleotide sequences that encode functional equivalents of the S avermitilis AveC gene product include naturally occurring aveC genes present in other strains of S avermitilis, aveC homolog genes present in other species of Streptomyces, and mutated aveC alleles, whether naturally occurring or engineered
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an ammo acid sequence that is
• a "substantial portion" of the ammo acid sequence of FIGURE 1 means a polypeptide comprising at least about 70% of the ammo acid sequence shown in FIGURE 1 (SEQ ID NO 2), and that constitutes a functionally equivalent AveC gene product, as defined above
• the term "homologous” refers to a polypeptide encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or having the ammo acid sequence of FIGURE 1 (SEQ ID NO 2) but in which one or more ammo acid residues has been conservatively substituted with a different ammo acid residue, where such conservative substitution results in a functionally equivalent gene product, as defined above
• Conservative ammo acid substitutions are well-known in the art Rules for making such substitutions include those described by Dayhof, M D , 1978, Nat Biomed Res Found , Washington, D C , Vol 5, Sup 3, among others More specifically, conservative ammo acid substitutions are those that generally take place within a family of ammo acids that are related in the acidity, polarity, or bulkmess of their side chains Genetically encoded am o acids are
• an "AveC homolog gene product” is defined as a gene product having at least about 50% ammo acid sequence identity to an AveC gene product of S avermitilis comprising the ammo acid sequence encoded by the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the ammo acid sequence shown in FIGURE 1 (SEQ ID NO 2)
• the AveC homolog gene product is from S hygroscopicus, (described in EP
• a "substantial portion" of the ammo acid sequence of SEQ ID NO 4 means a polypeptide comprising at least about 70% of the ammo acid sequence of SEQ ID NO 4, and that constitutes a functionally equivalent AveC homolog gene product
• AveC homolog gene product is defined as a gene product that, when expressed in S hygroscopicus strain FERM BP-1901 in which the native aveC homolog allele has been inactivated, results in the production of substantially the same ratio and amount of milbemycins as produced by S hygroscopicus strain FERM BP-1901 expressing instead only the wild-type, functional aveC homolog allele native to S hygroscopicus strain FERM BP-1901
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S hygroscopicus nucleotide sequence of SEQ ID NO 3
• the term "homologous" when used to refer to a polynucleotide molecule comprising a nucleotide sequence that is homologous to the S hygroscopicus AveC homolog gene product-encoding sequence of SEQ ID NO 3 means a polynucleotide molecule having a nucleotide sequence (a) that encodes the same gene product as the nucleotide sequence of SEQ ID NO 3, or a substantial portion thereof, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code, or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the ammo acid sequence of SEQ ID NO 4, under moderately stringent conditions,
• AveC homolog gene product 14 and washing in 0.1xSSC/0.1 % SDS at 68°C (Ausubel et al., 1989, above), and encodes a functionally equivalent AveC homolog gene product as defined above.
• the present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the S. hygroscopicus AveC homolog gene product.
• a polypeptide that is homologous to the S. hygroscopicus AveC homolog gene product As used herein to refer to polypeptides that are homologous to the AveC homolog gene product of SEQ ID NO:4 from S. hygroscopicus, the term "homologous" means a polypeptide having the amino acid sequence of SEQ ID NO:4, but in which one or more amino acid residues has been conservatively substituted with a different amino acid residue, where such conservative substitution results in a functionally equivalent AveC homolog gene product, as defined above.
• the present invention further provides oligonucleotides that hybridize to a polynucleotide molecule having the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide molecule having a nucleotide sequence which is the complement of the nucleotide sequence of FIGURE 1 (SEQ ID NO:1) or SEQ ID NO:3.
• Such oligonucleotides are at least about 10 nucleotides in length, and preferably from about 15 to about 30 nucleotides in length, and hybridize to one of the aforementioned polynucleotide molecules under highly stringent conditions, i.e., washing in 6xSSC/0.5% sodium pyrophosphate at ⁇ 37°C for ⁇ 14-base oligos, at ⁇ 48°C for ⁇ 17-base oligos, at ⁇ 55°C for -20- base oligos, and at ⁇ 60°C for ⁇ 23-base oligos.
• the oligonucleotides are complementary to a portion of one of the aforementioned polynucleotide molecules.
• oligonucleotides are useful for a variety of purposes including to encode or act as antisense molecules useful in gene regulation, or as primers in amplification of aveC- or aveC homolog-encoding polynucleotide molecules.
• Additional aveC homolog genes can be identified in other species or strains of Streptomyces by using the polynucleotide molecules or oligonucleotides disclosed herein in conjunction with known techniques.
• an oligonucleotide molecule comprising a portion of the S. avermitilis nucleotide sequence of FIGURE 1 (SEQ ID NO:1 ) or a portion of the S. hygroscopicus nucleotide sequence of SEQ ID NO:3 can be detectably labeled and used to screen a genomic library constructed from DNA derived from the organism of interest. The stringency of the hybridization conditions is selected based on the relationship of the reference organism, in this example S. avermitilis or S.
• oligonucleotides are well known to those of skill in the art, and such conditions will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived.
• Such oligonucleotides are well known to those of skill in the art, and such conditions will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived.
• telomere length 15 preferably at least about 15 nucleotides in length and include, e g , those described in the examples below
• Amplification of homolog genes can be carried out using these and other oligonucleotides by applying standard techniques such as the polymerase chain reaction (PCR), although other amplification techniques known in the art, e g , the ligase chain reaction, can also be used
• Clones identified as containing aveC homolog nucleotide sequences can be tested for their ability to encode a functional AveC homolog gene product
• the clones can be subjected to sequence analysis in order to identify a suitable reading frame, as well as initiation and termination signals
• the cloned DNA sequence can be inserted into an appropriate expression vector, i ⁇ , a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence
• an appropriate expression vector i ⁇
• a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence Any of a variety of host/vector systems can be used as described below, including but not limited to bacterial systems such as plasmid, bacte ⁇ ophage, or cosmid expression vectors
• Appropriate host cells transformed with such vectors comprising potential aveC homolog coding sequences can then be analyzed for AveC-type activity using methods such as HPLC analysis of fermentation products, as described, e g , in
• the present invention further provides recombinant cloning vectors and expression vectors which are useful in cloning or expressing polynucleotide molecules of the present invention comprising, e g , the aveC ORF of S avermitilis or any aveC homolog ORFs
• the present invention provides plasmid pSE186 (ATCC 209604), which comprises the complete ORF of the aveC gene of S avermitilis All of the following description regarding the aveC ORF from S avermitilis, or a polynucleotide molecule comprising the aveC ORF from S avermitilis or portion thereof, or an S avermitilis AveC gene product, also refers to aveC homologs and AveC homolog gene products, unless indicated explicitly or by context
• Recombinant vectors of the present invention are preferably constructed so that the coding sequence for the polynucleotide molecule of the invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide
• regulatory element includes but is not limited to nucleotide sequences that encode mducible and non-mducible promoters, enhancers, operators and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences
• the coding sequence is in "operative association" with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both
• Typical plasmid vectors that can be engineered to contain a polynucleotide molecule of the present invention include pCR-Blunt, pCR2 1 (Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3 (Vara et al , 1989, J Bact 171 5872-5881), among many others
• transc ⁇ ptional regulatory regions or promoters for bacteria include the ⁇ -gal promoter, the T7 promoter, the TAC promoter, ⁇ left and right promoters, trp and lac promoters, trp-lac fusion promoters and, more specifically for Streptomyces, the promoters ermE, melC, and tipA, etc
• an expression vector was generated that contained the aveC ORF cloned adjacent to the strong constitutive ermE promoter from Saccharopolyspora erythraea The vector was transformed into S avermitilis, and subsequent HPLC analysis of fermentation products indicated an increased titer of avermectins produced compared to production by the same strain but which instead expresses the wild-type ave
• Fusion protein expression vectors can be used to express an AveC gene product- fusion protein
• the purified fusion protein can be used to raise antisera against the AveC gene product, to study the biochemical properties of the AveC gene product, to engineer AveC fusion proteins with different biochemical activities, or to aid in the identification or purification of the expressed AveC gene product
• Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode ⁇ -galactosidase and trpE fusions, maltose-binding protein fusions, glutathione-S-transferase fusions and polyhistidine fusions (carrier regions)
• an AveC gene product or a portion thereof can be fused to an AveC homolog gene product, or portion thereof, derived from another species or strain of Streptomyces, such as, e g , S hygroscopicus or S griseochromogenes In a particular embodiment described in Section 12, below, and depicted in FIGURE
• AveC fusion proteins can be engineered to comprise a region useful for purification
• AveC-maltose-binding protein fusions can be purified using amylose resin
• AveC-glutath ⁇ one-S-transferase fusion proteins can be purified using glutathione-agarose beads, and AveC-polyhistidme fusions can be purified using divalent nickel resin Alternatively, antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein
• a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to the AveC polypeptide
• a nucleotide sequence coding for the FLAGTM epitope tag International Biotechnologies Inc ), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding, e g , to the carboxyl terminus of the AveC polypeptide
• the expressed AveC polypeptide-FLAGTM epitope fusion product can then be detected and affinity-purified using commercial
• the expression vector encoding the AveC fusion protein can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed AveC polypeptide can be released from the carrier region or fusion partner by treatment with a specific protease
• the fusion protein vector can include DNA sequences encoding thrombm or factor Xa cleavage sites, among others
• a signal sequence upstream from, and in reading frame with, the aveC ORF can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed gene product
• signal sequences include those from ⁇ -factor, immunoglobulins, outer membrane proteins, penicillmase, and T-cell receptors, among others
• the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker
• a coding sequence is preferably in operative association with the regulatory element coding sequences, as described above
• Reporter genes that are useful in the invention are well- known in the art and include those encoding green fluorescent protein, luciferase, xylE, and tyrosmase, among others
• Nucleotide sequences encoding selectable markers are well- known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement Examples of such sequences include those that encode resistance to erythromycin, thiostrepton or kanamycin, among many others
• the present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the invention, and novel strains or cell lines derived therefrom
• Host cells useful in the practice of the invention are preferably Streptomyces cells, although other prokaryotic cells or eukaryotic cells can also be used
• Such transformed host cells typically include but are not limited to microorganisms, such as bacteria transformed with recombinant bacte ⁇ ophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with recombinant vectors, among others
• the polynucleotide molecules of the present invention are intended to function in Streptomyces cells, but can also be transformed into other bacterial or eukaryotic cells, e g , for cloning or expression purposes
• a strain of E coli can typically be used, such as, e g , the DH5 ⁇ strain, available from the American Type Culture Collection (ATCC), Rockville, MD, USA (Accession No 31343), and from commercial sources (Stratagene)
• Preferred eukaryotic host cells include yeast cells, although mammalian cells or insect cells can also be utilized effectively
• the recombinant expression vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous culture of cells
• the expression vector is generally introduced into host cells in accordance with known techniques, such as, e g , by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, e g , antibiotic resistance, associated with the recombinant vector, as described above
• the integration and maintenance of the aveC coding sequence either in the host cell chromosome or episomally can be confirmed by standard techniques, e g , by Southern hybridization analysis, restriction enzyme analysis, PCR analysis, including reverse transc ⁇ ptase PCR (r
• AveC biological activity e g , the production of specific ratios and amounts of avermectins indicative of AveC activity in, e g , S avermitilis host cells
• the transformed host cell is clonally propagated, and the resulting cells can be grown under conditions conducive to the maximum production of the AveC gene product
• Such conditions typically include growing cells to high density
• appropriate induction conditions such as, e g , temperature shift, exhaustion of nutrients, addition of gratuitous mducers (e g , analogs of carbohydrates, such as isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic byproducts, or the like, are employed as needed to induce expression
• the expressed AveC gene product is retained inside the host cells, the cells are harvested and lysed, and the product isolated and purified from the lysate under extraction conditions known in the art to minimize protein degradation such as, e g , at 4°C, or in the presence of protease inhibitors, or both
• the exhausted nutrient medium can simply be collected and the product isolated therefrom
• the expressed AveC gene product can be isolated or substantially purified from cell lysates or culture medium, as appropriate, using standard methods, including but not limited to any combination of the following methods ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density cent ⁇ fugation, and affinity chromatography
• the expressed AveC gene product exhibits biological activity, increasing purity of the preparation can be monitored at each step of the purification procedure by use of an appropriate assay Whether or not the expressed AveC gene product exhibits biological activity, it can be detected as based, e g , on size, or re
• the present invention thus provides a recombmantly-expressed S avermitilis AveC gene product comprising the ammo acid sequence encoded by the AveC gene product- encoding sequence of plasmid pSE186 (ATCC 209604), or the ammo acid sequence of FIGURE 1 (SEQ ID NO 2) or a substantial portion thereof, and homologs thereof
• the present invention further provides a recombinantly-expressed S. hygroscopicus AveC homolog gene product comprising the amino acid sequence of SEQ ID NO:4 or a substantial portion thereof, and homologs thereof.
• the present invention further provides a method for producing an AveC gene product, comprising culturing a host cell transformed with a recombinant expression vector, said vector comprising a polynucleotide molecule having a nucleotide sequence encoding an AveC gene product, which polynucleotide molecule is in operative association with one or more regulatory elements that control expression of the polynucleotide molecule in the host cell, under conditions conducive to the production of the recombinant AveC gene product, and recovering the AveC gene product from the cell culture.
• the recombinantly expressed S. avermitilis AveC gene product is useful for a variety of purposes, including for screening compounds that alter AveC gene product function and thereby modulate avermectin biosynthesis, and for raising antibodies directed against the AveC gene product.
• an AveC gene product of sufficient purity can be characterized by standard methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, biological activity in producing appropriate products in the avermectin biosynthetic pathway, etc.
• the amino acid sequence of the AveC gene product can be determined using standard peptide sequencing techniques.
• the AveC gene product can be further characterized using hydrophilicity analysis (see, e.g., Hopp and Woods, 1981 , Proc. Natl. Acad. Sci. USA 78:3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions of the AveC gene product. Structural analysis can be carried out to identify regions of the AveC gene product that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol.
• the present invention provides a polynucleotide molecule comprising a nucleotide sequence that is otherwise the same as the S. avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604) or the nucleotide sequence of the aveC ORF of
• S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1), but that further comprises one or more mutations, so that cells of S. avermitilis strain ATCC 53692 in which the wild-type aveC allele has been inactivated and that express the polynucleotide molecule comprising the mutated nucleotide sequence produce a different ratio or amount of avermectins than are produced by cells of S. avermitilis strain ATCC 53692 that instead express only the wild-type aveC allele.
• such polynucleotide molecules can be used to produce novel strains of S. avermitilis that exhibit a detectable change in avermectin production compared to the same strain which instead expresses only the wild-type aveC allele.
• such polynucleotide molecules are useful to produce novel strains of S. avermitilis that produce avermectins in a reduced class 2:1 ratio compared to the same strain which instead expresses only the wild-type aveC allele.
• such polynucleotide molecules are useful to produce novel strains of S.
• avermitilis that produce increased levels of avermectins compared to the same strain which instead expresses only the wild-type aveC allele.
• polynucleotide molecules are useful to produce novel strains of S. avermitilis in which the aveC gene has been inactivated.
• Mutations to the aveC coding sequence include any mutations that introduce amino acid deletions, additions, or substitutions into the AveC gene product, or that result in truncation of the AveC gene product, or any combination thereof, and that produce the desired result.
• the present invention provides polynucleotide molecules comprising the AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or the nucleotide sequence of the aveC ORF of S. avermitilis as presented in FIGURE 1 (SEQ ID NO:1 ), but that further comprise one or more mutations that encode the substitution of an amino acid residue with a different amino acid residue at selected positions in the AveC gene product.
• substitutions can be carried out at any of amino acid positions 55, 138, 139, or 230, or some combination thereof.
• Mutations to the aveC coding sequence are carried out by any of a variety of known methods, including by use of error-prone PCR, or by cassette mutagenesis.
• oligonucleotide-directed mutagenesis can be employed to alter the aveC ORF sequence in a defined way such as, e.g., to introduce one or more restriction sites, or a termination codon, into specific regions within the aveC ORF sequence.
• Methods such as those described in U.S. Patent 5,605,793, which involve random fragmentation, repeated cycles of mutagenesis,
• nucleotide shuffling can also be used to generate large libraries of polynucleotides having nucleotide sequences encoding aveC mutations.
• Targeted mutations can be useful, particularly where they serve to alter one or more conserved amino acid residues in the AveC gene product.
• AveC gene products For example, a comparison of deduced amino acid sequences of AveC gene products and AveC homolog gene products from S. avermitilis (SEQ ID NO:2), S. griseochromogenes (SEQ ID NO:5), and S. hygroscopicus (SEQ ID NO:4), as presented in FIGURE 6, indicates sites of significant conservation of amino acid residues between these species.
• Targeted mutagenesis that leads to a change in one or more of these conserved amino acid residues can be particularly effective in producing novel mutant strains that exhibit desirable alterations in avermectin production.
• Random mutagenesis can also be useful, and can be carried out by exposing cells of S. avermitilis to ultraviolet radiation or x-rays, or to chemical mutagens such as N-methyl-N'- nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustards. See, e.g., Ausubel, 1989, above, for a review of mutagenesis techniques.
• mutated polynucleotide molecules are generated, they are screened to determine whether they can modulate avermectin biosynthesis in S. avermitilis.
• a polynucleotide molecule having a mutated nucleotide sequence is tested by complementing a strain of S. avermitilis in which the aveC gene has been inactivated to give an aveC negative (aveC) background.
• the mutated polynucleotide molecule is spliced into an expression plasmid in operative association with one or more regulatory elements, which plasmid also preferably comprises one or more drug resistance genes to allow for selection of transformed cells.
• This vector is then transformed into aveC host cells using known techniques, and transformed ceils are selected and cultured in appropriate fermentation media under conditions that permit or induce avermectin production. Fermentation products are then analyzed by HPLC to determine the ability of the mutated polynucleotide molecule to complement the host cell.
• mutated polynucleotide molecules capable of reducing the B2:B1 ratio of avermectins, including pSE188, pSE199, and pSE231 , are exemplified in Section 8.3, below.
• the present invention provides methods for identifying mutations of the aveC ORF of
• the present invention provides a method for identifying mutations of the aveC ORF capable of altering the class 2:1 ratio of avermectins produced, comprising: (a) determining the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis in
• step (b) determining the class 2 1 ratio of avermectins produced by cells of the same strain of S avermitilis as in step (a) but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO 1 ) or a nucleotide sequence that is homologous thereto, and (c) comparing the class 2 1 ratio of avermectins produced by the S avermitilis cells of step (a) to the class 2 1 ratio of avermectins produced by the S avermitilis cells of step (b), such that if the class 2 1 ratio of avermectins produced by the S avermitilis cells of step (a) is
• the present invention provides a method for identifying mutations of the aveC ORF or genetic constructs comprising the aveC ORF capable of altering the amount of avermectins produced, comprising (a) determining the amount of avermectins produced by cells of a strain of S avermitilis in which the native aveC allele has been inactivated, and into which a polynucleotide molecule comprising a nucleotide sequence encoding a mutated AveC gene product or comprising a genetic construct comprising a nucleotide sequence encoding an AveC gene product has been introduced and is being expressed, (b) determining the amount of avermectins produced by cells of the same strain of S avermitilis as in step (a) but which instead express only an aveC allele having the nucleotide sequence of the ORF of FIGURE 1 (SEQ ID NO 1) or a nucleotide sequence
• a mutated polynucleotide molecule that modulates avermectin production in a desirable direction the location of the mutation in the nucleotide sequence can be determined
• a polynucleotide molecule having a nucleotide sequence encoding a mutated AveC gene product can be isolated by PCR and subjected to DNA sequence analysis using known methods By comparing the DNA sequence of the mutated aveC allele to that of the wild-type aveC allele, the mutat ⁇ on(s) responsible for the alteration in avermectin production can be determined
• S avermitilis AveC gene products comprising either single ammo acid substitutions at any of residues 55 (S55F), 138 (S138T), 139 (A139T), or 230 (G230D), or a double substitution at positions 138 (S138T) and 139 (A139T), yielded
• the present invention further provides compositions for making novel strains of S avermitilis, the cells of which contain a mutated aveC allele that results in the alteration of avermectin production
• the present invention provides recombinant vectors that can be used to target any of the polynucleotide molecules comprising mutated nucleotide sequences of the present invention to the site of the aveC gene of the S avermitilis chromosome to either insert into or replace the aveC ORF or a portion thereof by homologous recombination
• a polynucleotide molecule comprising a mutated nucleotide sequence of the present invention provided herewith can also function to modulate avermectin biosynthesis when inserted into the S avermitilis chromosome at a site other than at the aveC gene, or when maintained episomally in S avermitilis cells
• the present invention also provides
• Such gene replacement vectors can be constructed using mutated polynucleotide molecules present in expression vectors provided herewith such as pSE188, pSE199, and pSE231 , which expression vectors are exemplified in Section 8.3 below.
• the present invention provides vectors that can be used to insert a mutated aveC allele into cells of a strain of S. avermitilis to generate novel strains of cells that produce altered amounts of avermectins compared to cells of the same strain which instead express only the wild-type aveC allele.
• the amount of avermectins produced by the cells is increased.
• such a vector further comprises a strong promoter as known in the art, such as, e.g., the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situated upstream from, and in operative association with, the aveC ORF.
• a strong promoter as known in the art, such as, e.g., the strong constitutive ermE promoter from Saccharopolyspora erythraea, that is situated upstream from, and in operative association with, the aveC ORF.
• a vector can be plasmid pSE189, described in Example 11 below, or can be constructed by using the mutated aveC allele of plasmid pSE189.
• the present invention provides gene replacement vectors that are useful to inactivate the aveC gene in a wild-type strain of S. avermitilis.
• gene replacement vectors can be constructed using the mutated polynucleotide molecule present in plasmid pSE180 (ATCC 209605), which is exemplified in Section 8.1 , below (FIGURE 3).
• the present invention further provides gene replacement vectors that comprise a polynucleotide molecule comprising or consisting of nucleotide sequences that naturally flank the aveC gene in situ in the S.
• avermitilis chromosome including, e.g., those flanking nucleotide sequences shown in FIGURE 1 (SEQ ID NO:1 ), which vectors can be used to delete the S. avermitilis aveC ORF.
• the present invention further provides methods for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce an altered ratio and/or amount of avermectins compared to cells of the same strain of S. avermitilis that instead express only the wild-type aveC allele.
• the present invention provides a method for making novel strains of S. avermitilis comprising cells that express a mutated aveC allele and that produce an altered class 2:1 ratio of avermectins compared to cells of the same strain of S. avermitilis that instead express only a wild-type aveC allele, comprising transforming cells of a strain of S.
• avermitilis with a vector that carries a mutated aveC allele that encodes a gene product that alters the class 2:1 ratio of avermectins produced by cells of a strain of S. avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered class 2:1 ratio compared to
• the class 2 1 ratio produced by cells of the strain that instead express only the wild-type aveC allele In a preferred embodiment, the altered class 2 1 ratio of avermectins is reduced
• the present invention provides a method for making novel strains of S avermitilis comprising cells that produce altered amounts of avermectin, comprising transforming ceils of a strain of S avermitilis with a vector that carries a mutated aveC allele or a genetic construct comprising the aveC allele, the expression of which results in an alteration in the amount of avermectins produced by cells of a strain of S avermitilis expressing the mutated aveC allele or genetic construct as compared to cells of the same strain that instead express only the wild-type aveC allele, and selecting transformed cells that produce avermectins in an altered amount compared to the amount of avermectins produced by cells of the strain that instead express only the wild-type aveC allele
• the amount of avermectins produced in the transformed cells is increased
• the present invention provides a method for making novel strains of S avermitilis, the cells of which comprise an inactivated aveC allele, comprising transforming cells of a strain of S avermitilis that express a wild-type aveC allele with a vector that inactivates the aveC allele, and selecting transformed cells in which the aveC allele has been inactivated
• cells of a strain of S avermitilis are transformed with a gene replacement vector that carries an aveC allele that has been inactivated by mutation or by replacement of a portion of the aveC allele with a heterologous gene sequence, and transformed cells in which the native aveC allele of the cells has been replaced with the inactivated aveC allele are selected
• Inactivation of the aveC allele can be determined by HPLC analysis of fermentation products, as described below In a specific, though non-limiting, embodiment described in Section 8
• the present invention further provides novel strains of S avermitilis comprising cells that have been transformed with any of the polynucleotide molecules or vectors of the present invention
• the present invention provides novel strains of S avermitilis comprising cells which express a mutated aveC allele in place of, or in addition to, the wild-type aveC allele, wherein the cells of the novel strain produce avermectins in an altered class 2 1 ratio compared to the class 2 1 ratio of avermectins produced by cells of the same strain that instead express only the wild-type aveC allele
• the altered class 2 1 ratio produced by the novel cells is reduced
• novel strains are
• the ratio of B2 B1 avermectins produced by cells of a novel S avermitilis strain of the present invention expressing a mutated aveC allele which reduces the ratio of class 2 1 avermectins produced is between less than 1 6 1 to about 0 1 , in a more preferred embodiment, the ratio is between about 1 1 to about 0 1 , and in the most preferred embodiment, the ratio is between about 0 84 1 to about 0 1
• novel cells of the present invention produce cyclohexyl B2 cyclohexyl B1 avermectins in a ratio of less than 1 6 1
• novel cells of the present invention produce cyclohexyl B2 cyclohexyl B1 avermectins in a ratio of less than 1 6 1
• the present invention provides novel strains of S avermitilis comprising cells which express a mutated aveC allele, or a genetic construct comprising an aveC allele, in place of, or in addition to, the wild-type aveC allele, wherein the cells of the novel strain produce an altered amount of avermectins compared to cells of the same strain that instead express only the wild-type aveC allele
• the novel strain produces an increased amount of avermectins
• the genetic construct further comprises a strong promoter, such as the strong constitutive ermE promoter from Saccharopolyspora erythraea, upstream from and in operative association with the aveC ORF
• the present invention provides novel strains of S avermitilis comprising cells in which the aveC gene has been inactivated
• Such strains are useful both for the different spectrum of avermectins that they produce compared to the wild- type strain, and in complementation screening assays as described herein, to determine whether targeted or random mutagenesis of the aveC gene affects avermectin production
• S avermitilis host cells were genetically engineered to contain an inactivated aveC gene
• strain SE180-11 described in the examples below, was generated using the gene replacement plasmid pSE180 (ATCC
• FIGURE 3 which was constructed to inactivate the S avermitilis aveC gene by insertion of the ermE resistance gene into the aveC coding region
• the present invention further provides recombmantly expressed, mutated S avermitilis AveC gene products encoded by any of the aforementioned polynucleotide molecules of the invention, and methods of preparing the same
• the present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S avermitilis, which cells express a mutated aveC allele that encodes a gene product that alters the class 2 1 ratio of avermectins produced by cells of a strain of S avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture
• the class 2 1 ratio of avermectins produced in the culture by cells expressing the mutated aveC allele is reduced This process provides increased efficiency in the production of commercially valuable avermectins such as doramectm
• the present invention further provides a process for producing avermectins, comprising culturing cells of a strain of S avermitilis, which cells express a mutated aveC allele or a genetic construct comprising an aveC allele that results in the production of an altered amount of avermectins produced by cells of a strain of S avermitilis expressing the mutated aveC allele or genetic construct compared to cells of the same strain which do not express the mutated aveC allele or genetic construct but instead express only the wild-type aveC allele, in culture media under conditions that permit or induce the production of avermectins therefrom, and recovering said avermectins from the culture
• the amount of avermectins produced in culture by cells expressing the mutated aveC allele or genetic construct is increased
• the present invention further provides a novel composition of avermectins produced by a strain of S avermitilis expressing a mutated aveC allele that encodes a gene product that reduces the class 2 1 ratio of avermectins produced by cells of a strain of S avermitilis expressing the mutated aveC allele compared to cells of the same strain that instead express only the wild-type aveC allele, wherein the avermectins in the novel composition are produced in a reduced class 2 1 ratio as compared to the class 2 1 ratio of avermectins produced by cells of the same strain of S avermitilis that instead express only the wild-type aveC allele
• the novel avermectin composition can be present as produced in exhausted fermentation culture fluid, or can be harvested therefrom
• the novel avermectin composition can be
• Avermectins are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and aca ⁇ cides
• Avermectin compounds produced according to the methods of the present invention are useful for any of these purposes
• avermectin compounds produced according to the present invention are useful to treat va ⁇ ous diseases or conditions in humans, particularly where those diseases or conditions are caused by parasitic infections, as known in the art See, e g , Ikeda and Omura, 1997, Chem Rev 97(7) 2591-2609 More particularly, avermectin compounds produced according to the present invention are effective in treating a variety of diseases or conditions caused by endoparasites, such as parasitic nematodes, which can infect humans, domestic animals, swine, sheep, poultry, horses or cattle More specifically, avermectin compounds produced according to the present invention are effective against nematodes that infect humans,
• the avermectin compounds produced according to the present invention are also useful in treating ectoparasitic infections including, e g , arthropod infestations of mammals and birds, caused by ticks, mites, lice, fleas, blowflies, biting insects, or migrating dipterous larvae that can affect cattle and horses, among others
• the avermectin compounds produced according to the present invention are also useful as insecticides against household pests such as, e , the cockroach, clothes moth, carpet beetle and the housefly among others, as well as insect pests of stored grain and of agricultural plants, which pests include spider mites, aphids, caterpillars, and orthopterans such as locusts, among others
• Animals that can be treated with the avermectin compounds produced according to the present invention include sheep, cattle, horses, deer, goats, swine, birds including poultry, and dogs and cats
• An avermectin compound produced according to the present invention is administered in a formulation appropriate to the specific intended use, the particular species
• an avermectin compound produced according to the present invention can be administered orally in the form of a capsule, bolus, tablet or liquid drench or, alternatively, can be administered as a pour-on, or by injection, or as an implant
• Such formulations are prepared in a conventional manner in accordance with standard veterinary practice
• capsules, boluses or tablets can be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier additionally containing a disintegrating agent and/or binder such as starch, lactose, talc, magnesium stearate, etc
• a drench formulation can be prepared by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent, etc
• Injectable formulations can be prepared in the form of a sterile solution which can contain other substances such as, e g , sufficient salts and/or glucose to make the solution isotonic with blood
• Such formulations will vary with regard to the weight of active compound depending on the patient, or species of host animal to be treated, the severity and type of infection, and the body weight of the host Generally, for oral administration a dose of active compound of from about 0 001 to 10 mg per kg of patient or animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory However, there can be instances where higher or lower dosage ranges are indicated, as determined, e g , by a physician or veterinarian, as based on clinical symptoms
• an avermectin compound produced according to the present invention can be administered in combination with animal feedstuff, and for this purpose a concentrated feed additive or premix can be prepared for mixing with the normal animal feed
• an avermectin compound produced according to the present invention can be applied as a spray, dust, emulsion and the like in accordance with standard agricultural practice
• Streptomyces avermitilis ATCC 53692 was stored at -70°C as a whole broth prepared in seed medium consisting of Starch (Nadex, Lamg National) - 20g, Pharmamedia (Trader's
• EXAMPLE ISOLATION OF THE aveC GENE This example describes the isolation and characterization of a region of the Streptomyces avermitilis chromosome that encodes the AveC gene product As demonstrated below, the aveC gene was identified as capable of modifying the ratio of cyclohexyl-B2 to cyclohexyl-B1 (B2 B1) avermectins produced
• the myce a grown in the above medium were used to inoculate 10 ml of TSB medium (Difco Tryptic Soy Broth - 30 g, in 1 liter dH 2 0, autoclaved at 121°C for 25 m ) in a 25 mm x 150 mm tube which was maintained with shaking (300 rpm) at 28°C for 48-72 hrs 7.1.2.
• TSB medium Difco Tryptic Soy Broth - 30 g, in 1 liter dH 2 0, autoclaved at 121°C for 25 m
• 25 mm x 150 mm tube which was maintained with shaking (300 rpm) at 28°C for 48-72 hrs 7.1.2.
• E. coli A single transformed E coli colony was inoculated into 5 ml Lu ⁇ a-Bertani (LB) medium (Bacto-Tryptone - 10 g, Bacto-yeast extract - 5 g, and NaCI - 10 g in 1 liter dH 2 0, pH 7 0, autoclaved at 121°C for 25 m , and supplemented with 100 ⁇ g/ml ampicillin)
• the culture was incubated overnight, and a 1 ml aliquot placed in a 1 5 ml microcentrifuge tube
• the culture samples were loaded into the AutoGen 540TM automated nucleic acid isolation instrument and plasmid DNA was isolated using Cycle 3 (equipment software) according to manufacturer's instructions
• YEME medium contains per liter: Difco Yeast Extract - 3 g; Difco
• P buffer which contains: sucrose - 205 g; K 2 S0 4 - 0.25 g; MgCI 2 6H 2 0 - 2.02 g; H 2 0 - 600 ml; K 2 P0 4 (0.5%) - 10 ml; trace element solution * - 20 ml; CaCI 2 2H 2 0 (3.68%) - 100 ml; and MES buffer (1.0 M, pH 6.5) - 10 ml.
• Race element solution contains per liter: ZnCI 2 - 40 mg; FeCI 3 6H 2 0 - 200 mg; CuCI 2 2H 2 0 - 10 mg; MnCI 2 4H 2 0 - 10 mg; Na 2 B 4 0 7 10H 2 0 - 10 mg; (NH 4 ) 6 Mo 7 0 24 4H 2 0 - 10 mg).
• the pH was adjusted to 6.5, final volume was adjusted to 1 liter, and the medium was filtered hot through a 0.45 micron filter.
• the mycelia were pelleted at 3,000 rpm for 20 min, the supernatant was discarded, and the mycelia were resuspended in 20 ml P buffer containing 2 mg/ml lysozyme.
• the mycelia were incubated at 35°C for 15 min with shaking, and checked microscopically to determine extent of protoplast formation. When protoplast formation was complete, the protoplasts were centrifuged at 8,000 rpm for 10 min. The supernatant was removed and the protoplasts were resuspended in 10 ml P buffer.
• the protoplasts were centrifuged at 8,000 rpm for 10 min, the supernatant was removed, the protoplasts were resuspended in 2 ml P buffer, and approximately 1 x 10 9 protoplasts were distributed to 2.0 ml cryogenic vials (Nalgene).
• T buffer base contains: PEG-1000 (Sigma) - 25 g; sucrose - 2.5 g; H 2 0 - 83 ml.
• the pH was adjusted to 8.8 with 1 N NaOH (filter sterilized), and the T buffer base was filter-sterilized and stored at 4°C.
• Working T buffer made the same day used, was
• S lividans TK64 (provided by the John Innes Institute, Norwich, U K) was used for transformations in some cases Methods and compositions for growing, protoplastmg, and transforming S lividans are described in Hopwood ef al , 1985, Genetic Manipulation of Streptomyces, A Laboratory Manual, John Innes Foundation, Norwich, U K , and performed as described therein Plasmid DNA was isolated from S lividans transformants as described in Section 7 1 3, above
• the flask was incubated at 29°C for 12 days with shaking at 200 rpm After incubation, a 2 ml sample was withdrawn from the flask, diluted with 8 ml of methanol, mixed, and the mixture centrifuged at 1 ,250 x g for 10 mm to pellet debris The supernatant was then assayed by HPLC using a Beckman Ultrasphere ODS column (25 cm x 4 6 mm ID) with a flow rate of 0 75 ml/mm and detection by absorbance at 240 nm The mobile phase was 86/8 9/5 1 methanol/water/ acetonitrile
• E coli strain GM2163 obtained from Dr B J Bachmann, Curator, E coli Genetic Stock Center, Yale University
• E coli strain DM1 BBL
• S lividans strain TK64 Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products
• Transformants of S avermitilis strain 1100-SC38 containing pSE119 produced an altered ratio of avermectin cyclohexyl-B2 cyclohexyl-B1 of about 3 7 1 (TABLE
• a new plasmid designated as pSE118, was constructed as follows Approximately 5 ⁇ g of pSE66 was digested with Sph ⁇ and BamHI The reaction mixture was loaded on a 0 8% SeaPlaque GTG agarose gel (FMC BioProducts), a 2 8 Kb Sphl/Ba HI fragment was excised from the gel after electrophoresis, and the DNA was recovered from the gel using GELaseTM (Epicentre Technologies) using the Fast Protocol Approximately 5 ⁇ g of the shuttle vector pWHM3 was digested with Sph ⁇ and BamHI About 0 5 ⁇ g of the 2 8 Kb insert and 0 5 ⁇ g of digested pWHM3 were mixed together and incubated overnight with 1 unit of ligase (New England Biolabs) at 15°C in a total volume of 20 ⁇ l according to supplier's instructions After incubation, 5 ⁇ l of the ligation mixture was incubated at 70°C for 10 mm, cooled to
• Protoplasts of S avermitilis strain 1100-SC38 were transformed with pSE118 as above Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products Transformants of S avermitilis strain 1100-SC38 containing pSE118 were not altered in the ratios of avermectin cyclohexyl-B2 avermectin cyclohexyl-B1 compared to strain 1100-SC38 (TABLE 2)
• a ⁇ 1 2 Kb fragment containing the aveC ORF was isolated from S avermitilis chromosomal DNA by PCR amplification using primers designed on the basis of the aveC nucleotide sequence obtained above
• the PCR primers were supplied by Genosys
• the ⁇ ghtward primer was 5'-TCACGAAACCGGACACAC-3'
• the thermal profile of the first cycle was 95°C for 5 mm (denaturation step), 60°C for 2 mm (annealing step), and 72°C for 2 mm (extension step)
• the subsequent 24 cycles had a similar thermal profile except that the denaturation step was shortened to 45 sec and the annealing step was shortened to 1 mm
• the PCR product was electrophoresed in a 1% agarose gel and a single DNA band of
• ⁇ 1 2 Kb was detected This DNA was purified from the gel, and ligated with 25 ng of linearized, blunt pCR-Blunt vector (Invitrogen) in a 1 10 molar vector-to-insert ratio following manufacturer's instructions The ligation mixture was used to transform One ShotTM Competent E coli cells (Invitrogen) following manufacturer's instructions Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the ⁇ 1 2 Kb insert was confirmed by restriction analysis This plasmid was designated as pSE179
• the insert DNA from pSE179 was isolated by digestion with BamHI/ i al, separated by electrophoresis, purified from the gel, and ligated with shuttle vector pWHM3, which had also been digested with BamHl/Xba ⁇ , in a total DNA concentration of 1 ⁇ g in a 1 5 molar vector-to-insert ratio
• the ligation mixture was used to transform competent E coli DH5 ⁇ cells according to manufacturer's instructions Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the ⁇ 1 2 Kb insert was confirmed by restriction analysis
• This plasmid which was designated as pSE186 (FIGURE 2, ATCC 209604), was transformed into E coli DM1 , and plasmid DNA was isolated from ampicillin resistant transformants
• the 2.9 Kb pSE119 fragment was sequenced and a -0.9 Kb ORF was identified (FIGURE 1) (SEQ ID NO:1), which encompasses a Pst Sph fragment that had previously been mutated elsewhere to produce B2 products only (Ikeda ef al., 1995, above).
• FGSURE 1 SEQ ID NO:1
• a comparison of this ORF, or its corresponding deduced polypeptide, against known databases did not show any strong homology with known DNA or protein sequences.
• TABLE 2 presents the fermentation analysis of S. avermitilis strain 1100-SC38 transformed with various plasmids.
• AveC mutants containing inactivated aveC genes were constructed using several methods, as detailed below.
• PCR product obtained was 543 bp and, when digested with BspE1 , three fragments of 368 bp, 96 bp, and 79 bp were observed, indicating chromosomal integration of the inactivated aveC gene and loss of the free rephcon
• mutations in the aveC gene that change both (i) nt position 970 from G to A, which changes the ammo acid at position 256 from a glycine (G) to an aspartate (D), and (n) nt position 996 from T to C, which changes the ammo acid at position 275 from tyrosme (Y)
• the S avermitilis aveC mactivation mutant strains SE180-11 , SE184-1-13, SE185- 5a, and others provided herewith, provide screening tools to assess the impact of other mutations in the aveC gene pSE186, which contains a wild-type copy of the aveC gene, was transformed into E coli DM1 , and plasmid DNA was isolated from ampicillin resistant transformants This pSE186 DNA was used to transform protoplasts of S avermitilis strain
• S avermitilis strain SE180-11 containing an inactive aveC gene was complemented by transformation with a plasmid containing a functional aveC gene
• the 1 2 Kb ORF was isolated by PCR amplification using primers designed on the basis of the aveC nucleotide sequence
• the ⁇ ghtward primer was SEQ ID NO 6 and the leftward primer was SEQ ID NO 7 (see Section 7 1 10, above)
• the PCR and subclonmg conditions were as described in Section 7 1 10
• DNA sequence analysis of the 1 2 Kb ORF shows a mutation in the aveC gene that changes nt position 337 from C to T, which changes the ammo acid at position 55 from senne (S) to phenylalanme (F)
• the aveC gene containing the S55F mutation was subcloned into pWHM3 to produce a plasmid which was designated as pSE187, and which was used to transform protoplasts of S avermitilis strain SE180-11 Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined, and Th ⁇ o r Erase
• pSE186 was digested with EcoRI and cloned into pGEM3Zf (Promega) which had been digested with EcoRI
• This plasmid which was designated as pSE186a, was digested with Apa ⁇ and Kpn ⁇ , the DNA fragments separated on an agarose gel, and two fragments of -3 8 Kb and -0 4 Kb were purified from the gel
• the -1 2 Kb insert DNA from pSE186 was used as a PCR template to introduce a single base change at nt position 585
• the PCR primers were designed to introduce a mutation at nt position 585, and were supplied by Genosys Biotechnologies, Inc (Texas)
• the ⁇ ghtward PCR primer was 5'- GGGGGCGGGCCCGGGTGCG
• the -1 2 Kb insert DNA from pSE186 was used as a PCR template
• the PCR primers were designed to introduce mutations at nt positions 585 and 588, and were supplied by Genosys Biotechnologies, Inc (Texas)
• the ⁇ ghtward PCR primer was 5'- GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3' (SEQ ID NO 14), and the leftward PCR primer was 5'-GGAACATCACGGCATTCACC-3' (SEQ ID NO 15)
• the PCR reaction was performed using the conditions described immediately above in this Section A PCR product of 449 bp was digested with Apal and Kpnl to release a 254 bp fragment, which was resolved by electrophoresis and purified from the gel pSE186a was
• FIGURE 1 (SEQ ID NO 1) comprises four different GTG codons at bp positions 42, 174, 177 and 180 which are potential start sites
• This section describes the construction of multiple deletions of the 5' region of the aveC ORF (FIGURE 1 , SEQ ID NO 1) to help define which of these codons could function as start sites in the aveC ORF for protein expression
• Fragments of the aveC gene variously deleted at the 5' end were isolated from S avermitilis chromosomal DNA by PCR amplification
• the PCR primers were designed based on the aveC DNA sequence, and were supplied by Genosys Biotechnologies, Inc
• the ⁇ ghtward primers were 5'-AACCCATCCGAGCCGCTC-3' (SEQ ID N0 16) (D1F1), 5'- TCGGCCTGCCAACGAAC-3' (SEQ ID NO 17) (D1 F2), 5'-CCAACGAACGTGTAGTAG-3' (
• PCR products were separated by electrophoresis in a 1% agarose gel and single DNA bands of either -1 0 Kb or -1 1 Kb were detected
• the PCR products were purified from the gel and ligated with 25 ng of linearized pCR2 1 vector (Invitrogen) in a 1 10 molar vector- to-insert ratio following the manufacturer's instructions
• the ligation mixtures were used to transform One ShotTM Competent E coli cells (Invitrogen) following manufacturer's instructions
• Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis and DNA sequence analysis These plasmids were designated as pSE190 (obtained with primer D1 F1), pSE191 (obtained with primer D1 F2), pSE192 (obtained with primer D1 F3), and pSE193 (obtained with primer D2F2)
• the insert DNAs were each digested with BamHl/Xbal, separated by electrophoresis, purified from the gel, and separately ligated with shuttle vector pWHM3, which had been digested with BamHUXbal, in a total DNA concentration of 1 ⁇ g in a 1 5 molar vector-to-insert ratio
• the ligation mixtures were used to transform competent E coli DH5 ⁇ cells Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the insert was confirmed by restriction analysis These plasmids, which were designated as pSE194
• the present invention allows aveC homolog genes from other avermectin- or milbemycin-producmg species of Streptomyces to be identified and cloned
• a cosmid library of S hygroscopicus (FERM BP-1901) genomic DNA was hybridized with the
• DNA and ammo acid sequence analysis of the aveC homologs from S hygroscopicus and S griseochromogenes indicates that these regions share significant homology (-50% sequence identity at the ammo acid level) both to each other and to the S avermitilis aveC
• pSE34 is the shuttle vector pWHM3 having the 300 bp ermE promoter inserted as a KpnllBamHl fragment in the
• pSE186 was digested with BamHI and Hindlll, the digest resolved by electrophoresis, and the 1 2 Kb fragment was isolated from the agarose gel and ligated with pSE34 which had been digested with BamHI and Hindlll
• the ligation mixture was transformed into competent E coli DH5 cells according to manufacturer's instructions Plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the 1 2 Kb insert was confirmed by restriction analysis
• This plasmid which was designated as pSE189, was transformed into E coli DM1 , and plasmid DNA isolated from ampicillin resistant transformants
• Protoplasts of S avermitilis strain 1100-SC38 were transformed with pSE189 Thiostrepton resistant transformants of strain 1100-SC38 were isolated and analyzed by HPLC analysis of fermentation products
• S avermitilis strain 1100-SC38 transformants containing pSE189 were altered in the ratios of avermectin cyclohexyl-B2 avermectin cyclohexyl-B1 produced (about 3 1 ) compared to strain 1100-SC38 (about 34 1), and total avermectin production was increased approximately 2 4-fold compared to strain 1100-SC38 transformed with pSE119 (TABLE 5)
• 53 pSE189 was also transformed into protoplasts of a wild-type S. avermitilis strain. Thiostrepton resistant transformants were isolated and analyzed by HPLC analysis of fermentation products. Total avermectins produced by S. avermitilis wild-type transformed with pSE189 were increased approximately 2.2-fold compared to wild-type S. avermitilis transformed with pSE119 (TABLE 5).
• a hybrid plasmid designated as pSE350 was constructed that contains a 564 bp portion of the S. hygroscopicus aveC homolog replacing a 564 bp homologous portion of the
• S. avermitilis aveC ORF (FIGURE 7), as follows.
• pSE350 was constructed using a BsaAI restriction site that is conserved in both sequences (aveC position 225), and a Kpnl restriction site that is present in the S. avermitilis aveC gene (aveC position 810).
• the Kpnl site was introduced into the S. hygroscopicus DNA by PCR using the rightward primer 5'-
• CTTCAGGTGTACGTGTTCG-3' (SEQ ID NO:23) and the leftward primer 5'-
• GAACTGGTACCAGTGCCC-3' (SEQ ID NO:24) (supplied by Genosys Biotechnologies) using PCR conditions described in Section 7.1.10, above.
• the PCR product was digested with BsaAI and Kpnl, the fragments were separated by electrophoresis in a 1% agarose gel, and the 564 bp BsaMIKpnl fragment was isolated from the gel.
• pSE179 (described in Section
• hygroscopicus were ligated together in a 3-way ligation and the ligation mixture transformed into competent E. coli DH5 ⁇ cells. Plasmid DNA was isolated from ampicillin resistant transformants and the presence of the correct insert was confirmed by restriction analysis using Kpnl and >4val. This plasmid was digested with Hindlll and Xbal to release the 1.2 Kb insert, which was then ligated with pWHM3 which had been digested with Hindlll and Xbal. The ligation mixture was transformed into competent E. coli DH5 ⁇ cells, plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis using Hindlll and Aval This plasmid DNA was transformed into E.
• plasmid DNA was isolated from ampicillin resistant transformants, and the presence of the correct insert was confirmed by restriction analysis and DNA sequence analysis.
• This plasmid was designated as pSE350 and used to transform protoplasts of S. avermitilis strain SE180-11. Thiostrepton resistant transformants of strain SE180-11 were isolated, the presence of erythromycin resistance was determined and Thio r Erm r transformants were analyzed by HPLC analysis of fermentation products. Results show that transformants containing the S. avermitilis/S. hygroscopicus hybrid plasmid have an average B2:B1 ratio of about 109:1 (TABLE 6) .
• accession numbers The following biological material was deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 20852, USA, on January 29, 1998, and was assigned the following accession numbers:

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